Radford GroupProtein folding

Berry groupEnzymology

Research in The Berry Group

How to tailor enzyme activity

The group investigates the possibility of altering the properties of enzymes to carry out unnatural, but desirable, reactions using the methods
of protein engineering and directed evolution.
At the University of Leeds, our research includes the
important class of enzymes, the aldolases, which catalyse the formation of carbon-carbon bonds. This type of reaction lies at the heart of
synthetic organic chemistry and is crucially important both within the cell and in industry for increasing the molecular complexity of
biomolecules. Rationale redesign of the enzyme lead to changes in substrate specificity. More recently, it has become apparent that Man's
understanding of the subtleties of protein structure and its
impact on catalysis is limited, and new, more random approaches (termed Directed Evolution) that mimic natural evolution have been developed
to allow Nature to teach us how to create new enzymes. We have embraced this methodology and have had some notable successes in altering
the specificity, reaction chemistry and stability of enzymes using this approach.

Having used FBP-aldolases to develop the methodology of both rational design and directed evolution of enzyme catalysis, in 2006 I changed
direction to work on the synthetically important, N-acetylneuraminic acid lyase (NAL). This enzyme is responsible for the synthesis of
sialic acid, a molecule involved in a myriad of important cellular recognition events such as the interaction of the influenza virus with
human cells. The work reached an exciting phase when we started tackling the difficult challenge of the construction of enzymes for the
specific stereochemically controlled synthesis of either of a pair of stereoisomers of a product at will. We were able to demonstrate, for
the first time, the directed evolution of a pair of complementary stereospecific enzymes. Considering that this involves the alteration of
the stereochemistry of the enzyme reaction itself, this is a striking result. These results highlighted a crucial role for residue 167 in
determining stereoselectivity, since alteration to glycine conferred 4S-stereoselectivity on the enzyme, while alteration to valine resulted
in 4R-specificity. This has been followed up by a demonstration that the same amino acid substitutions can be 'transplanted' into enzymes
engineered to have other substrate specificity and that the changes result in the same alterations in stereochemistry in those enzymes as
well. These results thus open the door to the construction of tailor-made enzymes for the synthesis of a wide range of complex molecules
including antibiotics, antivirals and anti-cancer drugs. Such biocatalytic synthetic steps are new being seen as the way forward for the
construction of the next generation of drug molecules by the pharmaceutical industries. This work is now being extended to generate
fluorinated compounds and we are particularly interested in the possibility of engineering the aldol condensation to generate two
stereo-centres at will during the enzyme reaction.

To fully benefit from directed evolution experiments, we need to understand the underlying fundamental principles of the structure-activity
relationship in engineered enzymes. This is especially important in understanding how the alterations of substrate specificity, increases
in stability and enzyme reaction stereochemistry have been brought about by the small number of mutations introduced during
the directed evolution and their effect on the enzyme structure. I have therefore driven a new avenue of collaborative research initially
with Prof Simon Phillips, and since his departure from Leeds, with Dr Arwen Pearson. A jointly supervised PhD student has recently solved
the X-ray crystal structures of around 10 of the evolved enzymes and the secrets of how the changes have been brought about are now being
revealed. For example, the change of enzyme substrate specificity from the normal glycerol-based group at C6 of sialic acid to a
dipropylamide group seems to be mainly controlled by the size of the residue at position 192, and full libraries of variants at this position
have been constructed and characterised. Excitingly, these structures have also shed light on the mechanism of these enzymes, a hotly debated
topic, and we have developed a new strategy using disabled mutants of the enzyme (particularly at Tyr-137) to trap reaction intermediates
along the catalytic pathway to reveal the details of the full mechanism.

Another of my recent developments has been to extend the work described above to the subsequent enzymes from the biosynthetic pathways
of complex sialylated carbohydrates. Such carbohydrates mediate critical interactions in processes as diverse as protein folding,
protein trafficking, antigenicity, in vivo half-life, cell-cell signalling, inflammation, in oncogenic transformations and tumorogenesis
in vertebrates and in mechanisms of infection and immunity in microbe-host interactions. Sialylated complex carbohydrates are therefore
important target molecules, but their size and stereocomplexity means that they are very difficult to synthesise. We have cloned and
overexpressed a number of enzymes from the pathways of sialylated sugars from Campylobacter and Pasteurella including CMP-sialic acid
synthetase and the sialyl transferases, CstII and Pst. Directed evolution of these enzymes is well underway, and new assays are being
developed. These rely either on tracking pH changes associated with the enzyme reaction, on trapping fluorescent products in cells or on
a novel FRET-based assay. The required molecular biology is now complete and libraries of variants have been constructed.
Library screening is underway to search for variants with altered specificities. One of the new areas of research underway in this area
is to alter not only the substrate specificity and stereochemistry, but also the regiochemistry of these important reactions.
In the longer term, these experiments should lead to developments in metabolic engineering where simple feed-stuffs can be converted to
complex carbohydrates in multistep pathways in engineered bacteria.

Finally in this area, we have (since May 2009) started an exciting new line of research. The 20 canonical amino acids cannot always
provide enough functionality to allow catalysis of all the cell's required reactions, and Nature occasionally uses post-translational
modifications to provide new functionality. We, as protein engineers, now have the capablility of also incorporating non-proteogenic
amino acids into enzymes. We have adopted both the method of chemical modification and in vivo incorporation of unnatural amino acids
using orthogonal tRNA/tRNA-RS pairs and have demonstrated the ability to remove and then reconstruct enzyme activity in this way.
This opens an exciting possibility of further extending the range of enzyme catalysis to novel reaction types.

We would like to thank the following organisations for the funds to support our research over the years:
BBSRC, EPSRC, The Royal Society, The Wellcome Trust, and The Leverhulme Trust.